Detection of microscopic objects

ABSTRACT

A substrate for use in manufacture of a production master plate for production of a detection disc for carrying samples in an apparatus for detection of microscopic objects in a fluid, the substrate having a channel and separate focus structure, wherein the focus structure is a groove.

This application is a divisional application of U.S. patent applicationSer. No. 14/682,682 which was filed on Apr. 9, 2015. That application inturn claims the benefit of Provisional Application Ser. No. 61/979,319which was filed on Apr. 14, 2014. The entire contents of thoseapplications are incorporated hereinto by reference.

The present invention relates to detection of microscopic objects and torelated products, apparatuses and methods.

It is important in many fields to be able to detect and/or count smallobjects such as bioparticles, molecules, cells and so on. One particularfield where this is of use is in biotechnology, for example in relationto DNA replication/amplification, where it can be important to be ableto detect molecular elements such as rolling circle products (RCPs).

One known system for detecting microscopic objects is flow cytometry.This is a laser-based, biophysical technology employed in cell counting,cell sorting, biomarker detection and protein engineering. Cells aresuspended in a stream of fluid and passed by an electronic detectionapparatus. Flow cytometry allows simultaneous multiparametric analysisof the physical and chemical characteristics of up to thousands ofparticles per second.

However, flow cytometry is not able to detect individual small objectsat a microscopic scale, especially not in a sample of the type obtainedby RCP production.

Flow cytometry is an example of a method that measures the integratedfluorescence within a defined volume, rather than the specific objectsthat may be contained within the volume. A particular problem arises forthese methods when the label used for detection is also present insolution and not only associated with the object of interest. Thepresence of free labels in solution then easily overwhelms the signalfrom the object of interest. Another problem arises when the object ofinterest is very small, such as in single molecule detection methods. Akey element to success is to improve signal over background and this isusually performed by reducing the investigated volume by various means.If the volume is small (<1 fL), background can be reduced to a minimumsince it scales linearly with volume. The signal however does not changewith decreased volume and thus signal over background is increased. Thishowever causes an unreasonably long analysis time if a large volume,such as several microliters or even more several hundred of microlitersor even millilitres of sample is to be investigated. Another solution tothe problem is to image the fluorescence volume to be analysed with aresolution comparable to the size of objects to be detected. These canthen be identified in the image as small areas of locally higherfluorescence intensity.

There is hence a need for new products, methods and apparatuses thatwould enable the detection and/or counting of single small objects, forexample for single molecule detection in the context of RCPs.

Viewed from one aspect, the invention provides a substrate for use inmanufacture of a production master plate for production of a detectiondisc for carrying samples in an apparatus for detection of microscopicobjects in a fluid, the substrate having a channel and separate focusstructure, wherein the focus structure is a groove.

To produce a functional structure comprising both a focus structure anda flow structure (also referred to as a channel, a flow channel or adetection channel) using a replication process, first the desiredpattern may be transferred to a substrate, which may have acrystallographic structure. The substrate may comprise silicon. Thesubstrate when processed may then be used to serve as a template ofopposite polarity compared to a replication master (also referred to asa production master plate, or simply a master) used for production ofthe flow channel and focus structure. The replication process may forinstance be thermoplastic injection moulding or casting. The productionmaster plate may comprise nickel and may be produced by electroplating.The replication master may be used in CD-based thermoplastic injectionmoulding, to generate CD shaped substrates having the desired flowchannel and focus structure. The focus structure is a groove in that itmay have a cross-section that is constant along one primary direction ofthe focus structure, for example its length or width. The channel andgroove may be provided in the (100) surface of the silicon. The focusstructure is a groove in that it may have a cross-section that isconstant along one primary direction of the focus structure, for exampleits length or width. The focus structure may have sloping {111}-orientedsidewalls. The focus structure may have a V-shaped cross-section. Thefocus structure may be oriented at right angles (that is, at 90 degrees)to the (110) plane. The channel may have a square or rectangularcross-section. Alternatively, the channel may have a U-shaped crosssection, or may otherwise have a curved base such that the channel hasan arc-shaped, inverted-arch-shaped or semi-circular shapedcross-section. The depth of the focus structure may be less than thedepth of the channel. The depth of the focus structure may have a fixed(that is, predetermined, known in advance, or non-arbitrary)relationship with the depth of the channel. The depth of the channel maybe twice the depth of the focus structure. Here, the depth of each ofthe focus structure and channel is taken to be the maximum depth, incases where the depth varies across the cross-section of the focusstructure or channel.

There may be multiple detection channels and multiple focus structures.The multiple detection channels may be arranged radially about a discshape of the substrate, and wherein there may be at least one focusstructure associated with each detection channel. In the instances wherethere are several channels on one disc, the channels may have differentgeometries and/or depths. The depth of the focus structure may have apredetermined relationship with the depth of the channel such that thesame algorithms and optical settings can be used for every channel,independent of the geometry and/or depth of the channel. It is alsopossible to have independent or fixed offsets between the focal plane ofthe focus structure and the focal plane in the detection channel.

In another aspect, the invention provides a detection disc for carryingsamples in an apparatus for detection of microscopic objects in a fluid,the detection disc having a detection channel and separate focusstructure, wherein the focus structure is a groove, and wherein thedetection disc is made using a production master plate manufacturedusing a substrate as described above. The invention also provides adetection disc for carrying samples in an apparatus for detection ofmicroscopic objects in a fluid, the detection disc having a detectionchannel and separate focus structure, wherein the focus structure is agroove.

The disc may be made from a material with refractive index of greaterthan 1.22. The disc may be made from an optically transparent material.The disc may be made from a thermoplastic polymer. The disc may be madefrom a cyclo olefin polymer. The disc may be made from Zeonor® 1060R.The disc may be made from PDMS, UV-grade PMMA, PMMA, PC or a COCpolymer-based material.

The focus structure is a groove in that it may have a cross-section thatis constant along one primary direction of the focus structure, forexample its length or width. The focus structure may have slopingsidewalls. The focus structure may have a V-shaped cross-section. Thedetection channel may have a square or rectangular cross-section.Alternatively, the channel may have a U-shaped cross section, or mayotherwise have a curved base such that the channel has an arc-shaped,inverted-arch-shaped or semi-circular shaped cross-section. The depth ofthe focus structure may be less than the depth of the channel. The depthof the focus structure may have a fixed (that is, predetermined, knownin advance, or non-arbitrary) relationship with the depth of thechannel. The depth of the channel may be twice the depth of the focusstructure. Here, the depth of each of the focus structure and channel istaken to be the maximum depth, in cases where the depth varies acrossthe cross-section of the focus structure or channel.

The detection disc may have multiple detection channels and multiplefocus structures. The multiple detection channels may be arrangedradially about the disc, and wherein there may be at least one focusstructure associated with each detection channel. In the instances wherethere are several channels on one disc, the channels may have differentgeometries and/or depths.

Viewed from a further aspect, the invention provides a method ofmanufacturing a substrate for manufacturing a master for a disc forcarrying samples in an apparatus for detection of microscopic objects ina fluid, the substrate having a channel and separate focus structure,wherein the focus structure is a groove, the method comprising:providing the substrate having a crystallographic structure; forming thechannel in the substrate with an orientation independent of theorientation of the crystalline planes; and forming the focus structurein the substrate with an orientation aligned with one of the crystallineplanes.

The invention further extends to a method of manufacturing a master fora disc for carrying samples in an apparatus for detection of microscopicobjects in a fluid, wherein the method of manufacture includesmanufacturing a substrate as set out in the aspect described above, andusing the substrate to manufacture a master.

The master may be manufactured from the substrate by electroplating. Themaster may be made of nickel.

The invention further extends to a method of manufacturing a disc forcarrying samples in an apparatus for detection of microscopic objects ina fluid, wherein the method of manufacture includes manufacturing asubstrate as set out in the aspect described above, using the substrateto manufacture a master, and using the master to manufacture the disc.

The disc may be manufactured from the master by injection moulding orcasting.

The substrate may be silicon. The channel and focus structure may beetched into the (100) plane of the silicon. The focus structure may beetched along the {111} plane of the silicon. The focus structure mayhave a V-shaped cross-section. The channel may be etched to a depth D,and the focus structure may be etched to a depth that may be less thanD. The depth of the focus structure may have a fixed (that is,predetermined, known in advance, or non-arbitrary) relationship with thedepth of the channel. The focus structure may be etched to a depth ofD/2.

Formation of the channel may be achieved using for instance the Boschprocess or variants of the Bosch process, for example, DRIE dry etching.The focus structure may be formed using hydroxide etching, for instanceusing KOH, where, in silicon, the etch rate for the (110) plane isgreater than the etch rate for the (100) plane, which is greater thanthe etch rate for the (111) plane. Therefore, a focus structure alignedwith a crystalline axis can be formed.

Alternatively to KOH, TMAH (Tetramethylammonium hydroxide), EDP(Ethylenediamine Pyrocatechol), CsOH, NaOH or N₂H₄—H₂O (Hydrazone) maybe used. Depending on the desired depth of the focus structures anappropriate mask is chosen. For shallow structures (for example, withdepth of less than 50 μm) SiO₂ is usually the simplest to use. Forstructures deeper than 50 μm one could chose SiN_(x) as a mask.

The use of V-shaped focusing structures can be combined with rectangularshaped flow structures but it is not limited to be combined with thistype of geometry for the flow structures. It is also possible to use theV-shaped focusing structures with arc-shaped flow or control channelsand this could be beneficial especially if optical registration is totake place in the arch-shaped channel.

The substrate may be provided with features as described above.

A still further aspect provides a method of manufacturing a detectiondisc for carrying samples in an apparatus for detection of microscopicobjects in a fluid, the detection disc comprising injection moulding thedetection disc using a production master plate produced using thesubstrate as described above.

Another aspect provides a method of manufacturing a detection disc forcarrying samples in an apparatus for detection of microscopic objects ina fluid, the method comprising: providing a substrate having acrystallographic substrate; forming the channel in the substrate with anorientation independent of the orientation of the crystalline planes;forming the focus structure in the substrate with an orientation alignedwith one of the crystalline planes; using the substrate to produce aproduction plate; and using the production master plate to manufacturethe detection disc having a detection channel and a focus structure.

The master may be produced by electroplating using the substrate as atemplate. The master may be made of nickel. The method may includeforming a mould from the master; and injection moulding the detectiondisc using the mould in order to obtain a detection disc having adetection channel and a focus structure.

The focus structure may be formed to have a predetermined depth inrelation to the depth of the channel. For example, the focus structuremay have a depth that is related to the depth of the channel by aspecific ratio.

This method may include providing the detection disc with features asdescribed above.

A yet further aspect provides a method of finding a focus plane in anapparatus for detection of microscopic objects in a fluid, wherein themethod comprises: providing an optically transparent substrate having afocus structure, the focus structure comprising a groove with slopingsidewalls provided in a first surface of the substrate; illuminating thesubstrate from a second surface, opposed to the first surface; imagingthe focus structure on the first surface using an imaging apparatus, andfocussing the imaging apparatus on the focus structure, wherein theangle between each sidewall of the focus structure and the first surfacemay be greater than the critical angle of the substrate.

The focus structure is a groove in that it may have a cross-section thatis constant along one primary direction of the focus structure, forexample its length or width. The focus structure may have a V-shapedcross-section. The method may include using information about the focusplane found by the imaging apparatus to focus a further, separate,imaging apparatus to the same focus plane. The focussing imagingapparatus may be an area detector and the further imaging apparatus maybe a line detector. The method may comprise illuminating the detectionchannel with a line beam, the projection of the line beam beingco-linear to the line of the line detector at the imaging plane.

The substrate may be a detection disc as described above and the focusstructure may be a focus structure of the disc.

A further aspect provides a method of focussing a first imagingapparatus to a focus plane in an apparatus for detection of microscopicobjects in a fluid, wherein the method comprises: imaging a focusstructure with a second imaging apparatus, focussing the second imagingapparatus on the focus structure to find the focal plane; and focussingthe first imaging apparatus to the focal plane to which the secondimaging apparatus is focussed. The first imaging apparatus may be a linedetector, and the second imaging apparatus may be an area detector.Alternatively, an area detector is used as the first imaging apparatuswhere a selected line or a selected subset of lines can be read out,e.g. a CMOS array. Alternatively, a single line detector serves thepurpose of both the first and second imaging apparatus. Alternatively, asingle imaging apparatus where a selected line or a selected subset oflines can be read out, e.g. a CMOS array, serves the purpose of both thefirst and second imaging apparatus.

The focus structure is a groove in that it may have a cross-section thatis constant along one primary direction of the focus structure, forexample its length or width. The focus structure may comprise a groovewith sloping sidewalls provided in a first surface of the substrate,wherein the angle between each sidewall of the focus structure and thefirst surface may be greater than the critical angle of the substrate.The method may comprise illuminating the detection channel with a linebeam, the projection of the line beam being co-linear to the line of theline detector at the imaging plane. The line of the line detector andthe projection of the line beam may both be perpendicular to thechannel. The method may comprise using a detection disc as describedabove, where the focus structure is a focus structure of the disc.

In another aspect, the invention provides an apparatus for detectingand/or counting microscopic objects comprising: a detection disccomprising a detection channel for carrying the microscopic objects in asample fluid and a separate focus structure associated with thedetection channel, a first imaging apparatus for finding a focal planeby focussing on the focus structure; a second imaging apparatus fordetecting and/or counting the objects in the detection channel, thesecond imaging apparatus being focussed to the focal plane usinginformation about the focal plane from the first imaging apparatus. Thefirst imaging apparatus may be a line detector, and the second imagingapparatus may be an area detector. Alternatively, an area detector isused as the first imaging apparatus where a selected line or a selectedsubset of lines can be read out, e.g. a CMOS architecture.Alternatively, a single line detector serves the purpose of both thefirst and second imaging apparatus. Alternatively, a single imagingapparatus where a selected line or a selected subset of lines can beread out, e.g. a CMOS array, serves the purpose of both the first andsecond imaging apparatus.

The focus structure is a groove in that it may have a cross-section thatis constant along one primary direction of the focus structure, forexample its length or width. The focus structure may comprise a groovewith sloping sidewalls provided in a first surface of the substrate, andwherein the angle between each sidewall of the focus structure and thefirst surface may be greater than the critical angle of the substrate.The apparatus may include a light source for illuminating the detectionchannel with a line beam, the projection of the line beam beingco-linear to the line of the line detector at the imaging plane. Theline of the line detector and the projection of the line beam may bothbe perpendicular to the channel. The detection disc may be as describedabove.

A further aspect of the invention provides an apparatus for detectingand/or counting microscopic objects, the apparatus comprising: adetection disc comprising a detection channel for carrying themicroscopic objects in a sample fluid and a separate focus structureassociated with the detection channel,

a first imaging apparatus for finding a focal plane by focussing on thefocus structure; a second imaging apparatus for counting the objects ina detection channel, the second imaging apparatus being focussed to thefocal plane using information about the focal plane from the firstimaging apparatus, wherein the focus structure comprises a groove withsloping sidewalls provided in a first surface of the detection disc, andwherein the angle between each sidewall of the focus structure and thefirst surface is greater than the critical angle of the substrate.The focus structure is a groove in that it may have a cross-section thatis constant along one primary direction of the focus structure, forexample its length or width. The focus structure may have a V-shapedcross-section. The apparatus may comprise a further, separate, imagingapparatus that receives information about the focal plane found by thefocussing imaging apparatus and uses this information to focus on thefocal plane. The focussing imaging apparatus may be an area detector andthe further imaging apparatus may be a line detector. Alternatively, anarea detector is used as the first imaging apparatus where a selectedline or a selected subset of lines can be read out, e.g. a CMOSarchitecture. Alternatively, a single line detector serves the purposeof both the first and second imaging apparatus. Alternatively, a singleimaging apparatus where a selected line or a selected subset of linescan be read out, e.g. a CMOS array, serves the purpose of both the firstand second imaging apparatus.

The apparatus may include a light source for illuminating the detectionchannel with a line beam, the projection of the line beam beingco-linear to the line of the line detector at the imaging plane. Theline of the line detector and the projection of the line beam may bothbe perpendicular to the channel.

The detection disc may be as described above.

A still further aspect of the invention provides a sample processingmodule for an apparatus for detecting and/or counting microscopicobjects, the sample processing module comprising: a carousel for holdinga plurality of sample containers; a number of stations for sampleprocessing steps including a filling station for filling the samplecontainers, a heating station for heating a sample in the samplecontainer; a cooling station for cooling the sample in the samplecontainer; an agitation station for agitating the sample in the samplecontainer; and a purging station for purging the sample from the samplecontainer and passing it to a detection module for analysis of thesample; wherein the heating and cooling stations act on the samplewithout contact with the sample.

The heating station may use infra-red or heated air to heat the sample.Each of the filling, heating, cooling and purging stations may actwithout contact between the stations and the sample container held inthe carousel.

The filling station may comprise a dispensing nozzle arranged to fillthe sample container without contact with the container or carousel. Thepurging station may comprise a compressed air source arranged to expelthe sample through a hole in the container without contact between thecompressed air source and the container or carousel.

The sample processing module may be used in conjunction with theapparatus of any of the other aspects described above.

Certain preferred embodiments are described below by way of example onlyand with reference to the accompanying figures in which:

FIG. 1 shows a part of a moulded disc with detection channels and focusstructures, the focus structures being shown with exaggerated size forillustrative purposes;

FIG. 2 shows a focus structure in close up view;

FIGS. 3A and 3B illustrate a light beam directed at the focus structureand the resultant reflection and refraction of light rays;

FIGS. 4A and 4B show a schematic and a photograph respectively with, onthe left, an out of focus image and, on the right, a focussed image ofthe focus structure;

FIGS. 5A and 5B show two schematic cross-sections with the detectionchannel and focus structures for two example depths of channel;

FIGS. 6A-6D illustrate images of objects in a detection channel, theimages taken with different line rates of a line camera in each of FIGS.6A, 6B, 6C and 6D;

FIGS. 7A and 7B respectively illustrate correct and incorrect matchingof the optical sampling volume to the channel geometry;

FIG. 8 shows flow rate of the sample fluid across the width of adetection channel;

FIG. 9 shows an example apparatus using high speed fluorescencedetection to detect objects in the detection channels of a disc of thetype shown in FIG. 1;

FIGS. 10A and 10B show non-contact heating used in a sample processingmodule of an example apparatus; and

FIG. 11 shows purging of a microfluidic reaction container in the sampleprocessing module.

FIG. 12 shows the relation between the substrate having acrystallographic structure and the production master.

This disclosure concerns an apparatus for detecting of and counting ofmicroscopic objects, such as particles (particularly bio-particles),cells, micro-organisms such as bacteria or molecules includingmacromolecules. The microscopic objects can include any object that issuitable small in size and can be detected based on optical methods, inparticular fluorescence of the object or of a fluorescent compositionapplied to the object. If the objects are translucent objects withoutscattering properties, such as RCPs, the objects may be 10 μm or less insize, perhaps 5 μm in size or smaller. The objects may have a largestdimension of 2 μm or less, perhaps 1.5 μm or less, 1 μm or less and insome cases 0.5 μm or less.

The example apparatus of the preferred embodiment uses optical elementsto detect small objects in a detection channel 4 of a sample substrate 2in the form of a disc 2. FIG. 1 shows a part of the disc 2. The disc 2has multiple such detection channels 4 and can be rotated to allowrepeated testing or different tests during a single test cycle. FIG. 1shows a disc with the same geometry for each channel 4 but the channelgeometry could be varied on a single disc to allow different tests to beperformed. The disc 2 also includes a focus structure 6, explained inmore detail below. The focus structure 6 is used to determine a focusdepth for best detection of small objects in a fluid in the detectingchannel 4. Each detection channel has adjacent focus structures 6.

The focussing uses a dedicated focussing camera and it is carried outautomatically as a part of the test cycle for the apparatus. Thefocussing camera may be a CCD area imaging chip. It is an area camera,i.e. a camera that captures a two dimensional image. Focussing may becarried out by a suitable algorithm. One algorithm is explained below.The focus information obtained by the focussing camera is used to setthe focus depth for a line camera that detects the small objects in thedetection channel 4. This line detector may be any suitable line camera,such as a CCD linear sensor and the use of the line detector isdiscussed in more detail below. The output from the line detector is animage allowing counting of the small objects of interest.Advantageously, by the use of the proposed focus structure and detectionchannel it becomes possible to automatically focus accurately onto atext volume that is small enough for single small objects, such assingle molecules to be detected and counted. The apparatus can hence beused for single molecule detection, for example in relation to rollingcircle products (RCPs) generated during DNA replication/amplification.

The processes used to generate the samples for the apparatus may beconventional process for production of samples of RCPs, bacteria, cells,macro-molecules or any other microscopic object requiring detectingand/or counting. Possible processes that might be used to providesamples for use with the proposed new apparatus are described in[earlier patent publications., Of special interest are processesgenerating Rolling Circle Products as a result of detection of nucleicacids, proteins, cellular or other macromolecular targets via the use ofso called Padlock probes, Proximity ligation, Proximity Extension ormethods for circularization of nucleic acids as described in e.g. U.S.Pat. Nos. 7,790,388, 8,053,188, 7,320,860, RE44265, U.S. Pat. Nos.7,074,564, 5,871,921, 6,558,928, 8,664,164, US20140030721 orWO2012160083. One example process that could be used to generate samplesin the form of RCPs is described in the applicant's co-pending patentapplication GB 1321123.0. Fluorescent labelling of bacteria is wellknown in the art. Examples include the use of non-specific detectionprotocols that employ signal producing systems that stain the doublestranded DNA of bacteria, e.g., via intercalation. Representativedetectable molecules that find use in such embodiments includefluorescent nucleic acid stains, such as phenanthridinium dyes,including monomers or homo- or heterodimers thereof, that give anenhanced fluorescence when complexed with nucleic acids. Examples ofphenanthridinium dyes include ethidium homodimer, ethidium bromide,propidium iodide, and other alkyl-substituted phenanthridinium dyes. Thenucleic acid stain may be or may incorporate an acridine dye, or a homo-or heterodimer thereof, such as acridine orange, acridine homodimer,ethidium-acridine heterodimer, or 9-amino-6-chloro-2-methoxyacridine.The nucleic acid stain may be an indole or imidazole dye, such asHoechst 33258, Hoechst 33342, Hoechst 34580 (BIOPROBES 34, MolecularProbes, Inc. Eugene, Oreg., (May 2000)) DAPI(4′,6-diamidino-2-phenylindole) or DIPI(4′,6-(diimidazolin-2-yl)-2-phenylindole). Other permitted nucleic acidstains include, but are not limited to, 7-aminoactinomycin D,hydroxystilbamidine, LDS 751, selected psoralens (furocoumarins), styryldyes, metal complexes such as ruthenium complexes, and transition metalcomplexes (incorporating Tb3+ and Eu3+, for example). The nucleic acidstain may be a cyanine dye or a homo- or heterodimer of a cyanine dyethat gives an enhanced fluorescence when associated with nucleic acids.Any of the dyes described in U.S. Pat. No. 4,883,867 to Lee (1989), U.S.Pat. No. 5,582,977 to Yue et al. (1996), U.S. Pat. No. 5,321,130 to Yueet al. (1994), and U.S. Pat. No. 5,410,030 to Yue et al. (1995) (allfour patents incorporated by reference) may be used, including nucleicacid stains commercially available under the trademarks TOTO, BOBO,POPO, YOYO, TO-PRO, BO-PRO, PO-PRO and YO-PRO from Molecular Probes,Inc., Eugene, Oreg. Any of the dyes described in U.S. Pat. No. 5,436,134to Haugland et al. (1995), U.S. Pat. No. 5,658,751 to Yue et al. (1997),and U.S. Pat. No. 5,863,753 to Haugland et at (1999) (all three patentsincorporated by reference) may be used, including nucleic acid stainscommercially available under the trademarks SYBR, SYTO, SYTOX,PICOGREEN, OLIGREEN, and RIBOGREEN from Molecular Probes, Inc., Eugene,Oreg. The nucleic acid stain may be a monomeric, homodimeric orheterodimeric cyanine dye that incorporates an aza- orpolyazabenzazolium heterocycle, such as an azabenzoxazole,azabenzimidazole, or azabenzothiazole, that gives an enhancedfluorescence when associated with nucleic acids, including nucleic acidstains commercially available under the trademarks SYTO, SYTOX, JOJO,JO-PRO, LOLO, LO-PRO from Molecular Probes, Inc., Eugene, Oreg.

Other methods of staining bacteria utilizes stains that distinguish livefrom dead bacteria such as is described in e.g. U.S. Pat. No. 5,534,416or WO 2012092238.

Advantageously, the samples may be processed automatically using asample preparation module (SPM) provided as part of the detectionapparatus. The SPM may automatically prepare the samples as discussed inmore detail below.

Focus Structure

The focus structure 6 is placed just beside the detection channel 4 andis used as a surrogate marker. A dedicated focusing camera focusses onthe focus structure 6 instead of focusing directly on the objects to bedetected. An advantage of this is that no fluorescence signal is neededfor focusing, focusing is not dependent on the objects being in a fixedposition, and focusing is always made at a fixed depth, defined by thegeometry and depth of the focus structure 6. The depth of the focusmarker can be placed at any depth in relation to the depth of thedetection channel, as will be seen from the discussion below the focusmarker may have a depth that is a half of the depth of the detectionchannel.

The focus structure consists of a V-shaped groove in the moulded disc.The disc is manufactured by injection moulding using a production master(reference numeral 3 in FIG. 12) in e.g. nickel produced from a siliconsubstrate (a wafer of silicon, reference numeral 1 in FIG. 12). Thisprocess is similar to the processes used for manufacture of compactdiscs as used for storage of data and music. In fact a CD mouldingmachine may be used to create the discs.

To produce a functional structure comprising both a focus structure anda flow structure using a replication process, first the desired patternis transferred to a substrate having a crystallographic structure. Thesubstrate may comprise silicon. The substrate when processed is thenused to serve as a template of opposite polarity as a replication master(production master plate, or master) used for production of the flow andfocus structures. The replication process can for instance bethermoplastic injection moulding or casting. The production master platemay comprise nickel and may be produced by electroplating. In onepreferred embodiment the replication master is used in CD-basedthermoplastic injection moulding, to generate CD shaped substrateshaving the desired flow and focus structures as described above.

In the silicon substrate, the shape of the flow channels (also referredto as detection channels) are etched independent of the orientation ofthe crystal axes in the silicon substrate using deep reactive-ionetching (DRIE). The shape of the focus structure (V-groove) is etchedalong the (111) plane of the crystal wafer. The angle between thesurface of the master and the (111) plane is then 54.7 degrees.

The focus structure is etched by wet etching as described above, withe.g. potassium hydroxide (KOH). The depth of the focusing structure isdefined by the diameter of opening in the mask used for wet etching,such that it possible to define structures of different depth and tocorrelate those with the depth of the flow channels. The depth of thefocussing structure is set to be less that the depth of the flowchannel. In the examples shown in FIGS. 5A and 5B, the depth of the flowchannel is h, and the depth of the focus structure is h/2 (i.e. half thedepth of the flow channel). The same focusing strategy can then be usedindependent of the depth of the flow channel. The focussing camera canobtain good information about the focus depth even if the height andposition of the disc is not constant for different discs or fordifferent detection channels on the same disc. There is always a focusreference point adjacent to the detection channel that provides anaccurate focus depth for the channel of interest. It should be notedthat the relationship between focus structure depth and the detectionchannel depth can be set at a predetermined off-set to each other.

Since the silicon plane is used to wet-etch the focus structures theycan only be oriented in 90 degree angles from the top plane. This is notthe case for the DRIE etched flow channels, which are placed radiallyalong the disc. The combination of the two etching techniques generatesan optimal system to deliver both a focusing structure defined by thecrystal plane and an arbitrarily-oriented but rectangular-shaped flowchannel.

The materials used to manufacture the injection-moulded disc may beZeonor® 1060R with a refractive index of 1.5, or PDMS (refractive index1.4). The refractive index of the disc material should be greater thanabout 1.22 (1/sin(54.7)), for reasons explained below. Other suitablematerials can of course also be used for the detection disc such asUV-grade-PMMA, PMMA, PC or other COC polymer based materials. In orderto have as sharp focusing structures as possible it is preferable to usethermoplastic materials with excellent form-filling and flowcharacteristics.

When focusing, the grove is illuminated from below with a light beam,close to parallel, as shown in FIG. 3A. As can be seen from FIG. 3B, theincident angle of a collimated light beam on the sidewalls of the groovegives rise to total reflection when the refractive index of the discmaterial is greater than about 1.22 (i.e. 1/sin(54.7), where 54.7 is theangle between the surface of the master and the (111) planes). In thecase of a less than perfectly collimated beam, the reflection may not betotal, but sufficient for contrast detection as detailed below.

As a result of the total internal reflection, when viewed from the top,the area of the groove appears dark. If the imaging camera is focusedexactly on the bottom level of the groove, where the sidewalls of theV-shaped groove meet, a bright line appears. The contrast between thisbright line and the dark surrounding changes rapidly with changing focusplane, allowing precise contrast-based focusing. In each of FIGS. 4A and4B (which show corresponding view, as a schematic in FIG. 4A, and as aphotograph in FIG. 4B), the figure on the left shows the view when theimaging camera is out of focus, and the figure on the right shows theview when the imaging camera is in focus.

Focusing Algorithm

The focus plane of the image used for autofocus is first placed at aknown position relative to the position of the bottom of the groove.That is, it is known that the groove will not be in focus, but theposition is close enough so the dark area of the entire groove can beidentified. Also, it is known whether the actual focus position is onthe far or near side of the groove bottom.

First the algorithm finds the dark area of the groove, and masks this.Then, using a pre-defined threshold, the algorithm tries to find alighter area contained within the dark area. If it succeeds, a contrastvalue is calculated. The algorithm then moves the focal plane a definedstep size closer to the groove bottom, the procedure is repeated, and aslong as the contrast value increased, the algorithm moves in the samedirection. When a contrast value smaller than the previous iscalculated, the direction of movement is reversed, and the step size ishalved. This continues until a max focus value using the smallest stepsize possible is reached. The groove bottom is then focused to withinless than 1 μm, or even within less than 0.5 μm. The invention thusprovides a production method and a design of a focus structure enablingmore precise focussing using a very simple algorithm than existing othermeans of producing a substrate to be used for autofocussing of a sample.One of the key features of the invention is the production of the focusstructure in the same material as the detection channel and possibly atthe same time. No additional work that can affect the positioning of thefocus structure in relation to the focus volume in the detection channelneeds to be performed afterwards. Such as is the case using e.g. metalmarkers attached to a surface as is used in existing solutions. Thepositioning within less than 1 μm of the wanted focus plane of thecurrent invention is superior in precision compared to current methodsrelying solely on reflection.

Line Detector

A sample volume in the detection channel is imaged with a line detector(a line camera having a single line of pixels).

The different images that would be obtained for a given line rate areshown in FIGS. 6A to 6D, which show in the top drawings the particlesmoving through the channel, and in the bottom drawings, the imageobtained by the line camera. The image obtained is built up of a largenumber of lines.

In FIG. 6A, the object is stationary, and all lines are identical. Whenthe object moves slowly (as in FIG. 6B), the line image will changeslowly. When the object moves in sync with the rate of line images taken(as in FIG. 6C), the frame accurately reproduces the object. When theobject moves faster (as in FIG. 6D), the objects appears compressed, inthe extreme case to single short lines.

The sample fluid that is pumped through the detection channel has aparabolic flow rate over the channel width (see FIG. 8). The line rateof the detector, while variable, is the same over the whole line image.It is therefore not possible to achieve a 1:1 ratio between flow rateand the line rate (as in FIG. 6C) over the entire channel width. Howeverthis is not necessary when counting the number of microscopic particles(for example, RCPs). By adjusting the flow and line rate it is possibleto compress the 2D image of a particle into one single exposure of theline detector. It will then become a line of a certain width. This isdone for a certain region of interest (ROI) of the channel, excludingthe areas adjacent to the channel sides.

The advantage of compressing the image in this way is that the intensityof the pixels containing then object is strongly elevated over thebackground compared to 1:1 imaging. Furthermore, the image analysisspeed increases. The image size needed to record a given volume of fluidalso decreases. Finally, only very large objects falling within the ROIwill cover more than 1 line, and can be readily rejected as artefacts.

Optical Sampling Volume

The optical sampling volume should be matched to the channel geometry.FIG. 7A illustrates a matched setup, in which the entirety of theoptical sampling volume is contained within the sample volume (i.e.within the cross-section of the detection channel). FIG. 7B illustratesa mismatch, wherein the optical sampling volume is not entirelycontained within the sample volume. This will lead to unwantedreflections and other artefacts relating to the channel top and bottombeing included in the optical observation volume.

It is also clear from FIGS. 7A and 7B that the effect of channel driftin the z-axis is minimized using a deeper channel as smaller drifts willstill keep the observation volume within the channel. If a shallowchannel is used, any drift will sooner place the observation volumeoutside the channel.

The voxel in which an RCP will be imaged as a point is limited by theoptical resolution in the axial direction, and (depending on pixel size,numerical aperture and magnification, and flow rate) either the opticallateral resolution, the pixel size, or the flow rate. First, if the flowrate is disregarded (or set to 0), the lateral resolution using a 40×,MN1.3 objective with an excitation wavelength of 532 nm is 0.23 μm, andfor a 20× NA 0.8 objective 0.38 μm, according to Equation 1.

$\begin{matrix}{{FWHM}_{{ili},{lateral}} = {0.51\frac{\lambda_{exc}}{NA}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$Here, NA is the numerical aperture of the objective and λ_(exc) is thewavelength of the excitation light.

In the image plane, the projected distance is 9.05 or 7.65 μm for 40 and20× objectives, respectively. The pixel width of the line detector is 14μm. A typical RCP has a diameter of 0.5 μm, but can be around 1 μm, ore.g. 1.5 μm. RCPs can also be of larger size, e.g. 10 μm, 5 μm or 2.5 μmif using additional amplification of the object on the primary RCP. RCPshave the unusual property compared to many other objects of similar sizethat they do not have scattering properties. This translates in theimage plane to 20 μm for a 40× and 10 μm for a 20× objective. Thus, whenimaging an RCP the lateral resolution is limited by the pixel size sothat a typical RCP covers about 2 pixels using 40× and 1 pixel using a20× objective.

Conversely, the pixel sizes projected onto the object plane are 0.35 and0.7 μm for the 40 and 20× objectives.

With regard to axial resolution, this is described by Equation 2

$\begin{matrix}{{FWHM}_{\det,{axial}} = \sqrt{( \frac{0.88 \cdot \lambda_{em}}{n - \sqrt{n^{2} - {NA}^{2}}} )^{2} + ( \frac{\sqrt{2} \cdot n \cdot {PH}}{NA} )^{2}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$Here, λ_(em) is the emission wavelength, PH is the object-side pinholediameter (in μm), n is the refractive index of the immersion liquid (thefluid in which the objects to be counted are carried) and NA is thenumerical aperture of the objective.

Here, the height of the CCD pixel line, as a virtual slit, is used aspinhole size, 28 μm. It should be noted that this value is higher thanthe pinhole diameter required for optimum confocality (pinhole size=1Airy unit), which is 20 μm for a 40× NA 1.3 objective, and 16 μm for a20× 0.8 NA objective. This is in line with the main purpose of thedesign, namely to minimize the influence if fluorescence related tounbound detection fluorophores present throughout the channel. In thepresent configuration, the 28 μm slit height represents a balancebetween background rejection and signal loss.

Using eq. 2, the axial FWHM is 2.4 μm and 7.8 μm for 40× NA 1.3 and 20×NA 0.8 um, respectively. Thus, using a 40× 1.3 NA objective a voxel inthe object plane is about 0.35×0.35×2.4 μm for the 40×, and 0.7×0.7×7.8μm for the 20× objective.

When using a channel of a fixed height, the choice of these objectivesaffects the properties of the analysis. With the 40× objective the voxeldepth is significantly smaller than for the 20×. Thus, a smaller portionof the channel depth is scanned.

When the focal plane is centred in the mid-channel, (see FIG. 8) theflow rate variation over the voxel height and artefacts related to thechannel top and bottom are minimal. This translates into a morehomogenous volume to be analysed, and in turn a greater countingprecision. Using the 20×, on the other hand results in a larger portionof the channel being scanned at the cost of a certain precision loss.

The flow rate in the system is adjustable between 5 and 50 μl/minute. Acommon value is 25 μl/min. Using a channel with a 60×400 μmcross-section, the average flow velocity is then 17400 μm/second. Theline rate of the cameras is 5 kHz. The average movement of the fluid perexposure is then about 3.5 μm. The pixel height projected onto theobject plane is 1.4 μm if a 20× objective is used. Therefore, a standardRCP of about 0.5 μm diameter will completely pass the detector line inone exposure, giving a 1D representation of the fluorescence intensityprofile. In the above calculation, an average speed across the channelis assumed, although this is of course a simplification.

High-Fluorescence Detection of RCPs

One use for the apparatus is detection of RCPs. Solutions containing thelabelled RCPs are analysed using a dedicated high-speed fluorescencedetection instrument as shown in FIG. 9. The sample solution is pushedthrough a flow channel with a cross section of 200×40 μm (W×H). The flowchannels are aligned radially on a CD-format plastic disc as describedabove, with appropriate optically clear lid and fluid interfaces,allowing rapid change of channel in case of a malfunction or clog.

Three lasers with wavelengths of 488 nm (Calypso, 100 mW, Cobolt AB,Solna, Sweden), 532 nm (Samba 300 mW, Cobolt AB, and 640 nm (Cube 640,40 mW, Coherent Inc, Santa Clara, Calif.), are collimated throughindividually focusable beam expanders, bringing the beam diameters up toabout 8 mm (1/e2). The beams are made collinear by a system of steerabledichroic and full mirrors. The collinear beams passed through a beamshaping lens, designed to produce in conjunction with a high numericalobjective (Zeiss Fluar 40×, NA 1.3, Carl Zeiss AB, Stockholm, Sweden), aline illumination profile across the interrogation volume of the flowchannel. Finally, just prior to the objective entrance pupil, the laserlight passes through a laser-pass dichroic mirror (Semrock Inc,Rochester, N.Y.).

Fluorescent light is emitted by RCPs pumped across the interrogationvolume using a syringe pump (Tecan XLP6000, Tecan Nordic AB, Mölndal,Sweden). The emitted wavelength(s) corresponds to the emission spectraof the fluorescent labels bound to RCPs. The emitted light is collectedthrough the objective is reflected or passes through the dichroicmirror, and is further collected by CCD line detectors (DALSA Spyder 3,1024 pixels, line rate 5 kHz, Parameter AB, Stockholm, Sweden). Dichroicmirrors and band-pass filters are used to direct the light from eachspecific fluorophore to a specific detector. A small portion of thefluorescent light is redirected by a beam sampler onto a CCD areadetector (μEye UI-1545LE-M, Parameter AB, Stockholm, Sweden) in order toallow for channel alignment and focusing.

From each detector the results of each sample run are registered as aseries of x-t images where each RCP is identified through imageanalysis. The image analysis consists of background subtraction, patternrecognition, pattern matching across detectors for multiply fluorescentobjects discrimination of RCPs and non-specific events, and RCPcounting. For each reagent, a threshold for the number of RCPs is set todesignate a positive sample.

It will of course be appreciated that this example method can be adaptedfor use with small objects other than RCPs. Possible small objects thatcan be detected and counted by the proposed apparatus are discussedabove.

Sample Preparation Module

The Sample Processing Module (SPM) in this example accepts as a sampleeither nucleic acid targets to be interrogated, or templated nucleicacid circles created via well-known methods such as immuno-RCA orproximity ligation, indicating target proteins. Of course, other smallobjects as mentioned above could be used as the sample without departingfrom the main concepts described herein.

The SPM comprises a carousel for holding the samples. The carousel isadvanced one step every minute and a new sample can be accepted asrapidly as every 4th minute. If a sample is to be processed, a samplecontainer (microfluidic reaction container, MRC) is inserted into thecarousel, and stepwise transported and processed through the circle.

At certain positions, reagents are dispensed, the MRC is heated, cooled,agitated or purged through a simple, integrated valve.

All reactions such as dispensing, heating, cooling, agitation, andpurging are done in a contact-free manner (as shown in FIGS. 10A, 10Band 11), and the only contact with the processed sample is at the veryend of the process where the sample is aspirated to a detection module(DM) for readout.

The described SPM has the following advantages:

-   -   1) sample throughput of 100+ samples per day without refill of        reagents or consumables;    -   2) rate of analysis up to 1 sample/5 minutes;    -   3) random access;    -   4) minimal risk of cross-contamination between samples; and    -   5) small footprint and low weight.

The process carried out by the SPM can be any known process forpreparation of samples for detection. Compared to known devices, the SPMdiffers in that the reactions and in particular the heating are carriedout without contact.

The following clauses set out features of the invention which may notpresently be claimed in this application, but which may form the basisfor future claims, amendment or divisional applications.

Several embodiments of the disclosure will be discussed in the numberedparagraphs entitled “Clauses” set forth below.

CLAUSES

-   1. A substrate for use in manufacture of a production master plate    for production of a detection disc for carrying samples in an    apparatus for detection of microscopic objects in a fluid, the    substrate having a channel and separate focus structure, wherein the    focus structure is a groove.-   2. A substrate according to clause 1, wherein the substrate    comprises a material having a crystallographic structure.-   3. A substrate according to clause 2, wherein the substrate    comprises silicon.-   4. A substrate according to clause 3, wherein the channel and groove    are provided in the (100) surface of the silicon.-   5. A substrate according to clause 4 wherein the focus structure has    sloping {111}-oriented sidewalls.-   6. A substrate according to any preceding clause wherein the focus    structure has a V-shaped cross-section.-   7. A substrate according to clause 4, 5 or 6 wherein the focus    structure is oriented at right angles to the (110) plane.-   8. A substrate according to any preceding clause, wherein the    channel has a square or rectangular cross-section.-   9. A substrate according to any preceding clause, wherein the    channel has a U-shaped cross section, or may otherwise have a curved    base such that the channel has an arc-shaped, inverted-arch-shaped    or semi-circular shaped cross-section.-   10. A substrate according to any preceding clause, wherein the depth    of the focus structure is less than the depth of the channel.-   11. A substrate according to any preceding clause, wherein the depth    of the focus structure is at a predetermined depth in relation to    the channel depth.-   12. A substrate according to any preceding clause, wherein the depth    of the channel is twice the depth of the focus structure.-   13. A substrate according to any preceding clause, comprising    multiple detection channels and multiple focus structures.-   14. A substrate according to clause 13, wherein the multiple    detection channels are arranged radially about a disc shape of the    substrate, and wherein there is at least one focus structure    associated with each detection channel.-   15. A detection disc for carrying samples in an apparatus for    detection of microscopic objects in a fluid, the detection disc    having a detection channel and separate focus structure, and wherein    the focus structure is a groove, wherein the detection disc is made    using a production master plate manufactured using the substrate of    any preceding clause.-   16. A detection disc for carrying samples in an apparatus for    detection of microscopic objects in a fluid, the detection disc    having a detection channel and separate focus structure, wherein the    focus structure is a groove.-   17. A detection disc according to clause 15 or 16, wherein the disc    is made from a material with refractive index of greater than 1.22.-   18. A detection disc according to any of clauses 15 to 17, wherein    the disc is made from an optically transparent material.-   19. A detection disc according to any of clauses 15 to 18, wherein    the disc is made from a thermoplastic polymer.-   20. A detection disc according to clause 19, wherein the disc is    made from a cyclo olefin polymer.-   21. A detection disc according to clause 20, wherein the disc is    made from Zeonor® 1060R.-   22. A detection disc according to any of clauses 15 to 19 wherein    the disc is made from PDMS, UV-grade PMMA, PMMA, PC or a COC    polymer-based material.-   23. A detection disc according to any of clauses 15 to 22 wherein    the focus structure has sloping sidewalls.-   24. A detection disc according to clause 23 wherein the focus    structure has a V-shaped cross-section.-   25. A detection disc according to clause any of clauses 15 to 24,    wherein the detection channel has a square or rectangular    cross-section.-   26. A detection disc according to clause any of clauses 15 to 24,    wherein the detection channel has a U-shaped cross section, or may    otherwise have a curved base such that the channel has an    arc-shaped, inverted-arch-shaped or semi-circular shaped    cross-section.-   27. A detection disc according to any of clauses 15 to 26, wherein    the depth of the focus structure is less than the depth of the    channel.-   28. A detection disc according to any of clauses 15 to 27, wherein    the depth of the channel is twice the depth of the focus structure.-   29. A detection disc according to any of clauses 15 to 28,    comprising multiple detection channels and multiple focus    structures.-   30. A detection disc according to clause 29, wherein the multiple    detection channels are arranged radially about the disc, and wherein    there is at least one focus structure associated with each detection    channel.-   31. A method of manufacturing a substrate for manufacturing a master    for a disc for carrying samples in an apparatus for detection of    microscopic objects in a fluid, the substrate having a channel and    separate focus structure, wherein the focus structure is a groove,    the method comprising:    -   providing the substrate having a crystallographic structure;    -   forming the channel in the substrate with an orientation        independent of the orientation of the crystalline planes; and    -   forming the focus structure in the substrate with an orientation        aligned with one of the crystalline planes.-   32. A method according to clause 31, wherein the substrate is    silicon.-   33. A method according to clause 32, wherein the channel and focus    structure are etched into the (100) plane of the silicon.-   34. A method according to clause 33 wherein the focus structure is    etched along the {111} plane of the silicon.-   35. A method according to any of clauses 31 to 34 wherein the focus    structure has a V-shaped cross-section.-   36. A method according to any of clauses 31 to 35, wherein the    channel is etched to a depth D, and the focus structure is etched to    a depth that is less than D.-   37. A method according to any of clauses 31 to 36, wherein the focus    structure is formed to have a predetermined depth in relation to the    depth of the channel.-   38. A method according to clause 36 or 37, wherein the focus    structure is etched to a depth of D/2.-   39. A method according to any of clauses 31 to 38, wherein the focus    structure is etched by wet KOH etching.-   40. A method according to any of clauses 31 to 39, wherein the    channel is etched by DRIE.-   41. A method according to any of clauses 31 to 40 comprising    providing the substrate with features as described in any of clauses    1 to 14.-   42. A method of manufacturing a detection disc for carrying samples    in an apparatus for detection of microscopic objects in a fluid, the    detection disc comprising injection moulding the detection disc    using a production master plate produced using the substrate    according to any of clauses 1 to 14.-   43. A method of manufacturing a detection disc for carrying samples    in an apparatus for detection of microscopic objects in a fluid, the    method comprising:    -   providing a substrate having a crystallographic substrate;    -   forming the channel in the substrate with an orientation        independent of the orientation of the crystalline planes; and    -   forming the focus structure in the substrate with an orientation        aligned with one of the crystalline planes;    -   using the substrate to produce a production master plate; and    -   using the production master plate to manufacture the detection        disc having a detection channel and a focus structure.-   44. A method according to clause 42 or 43, wherein the disc is    formed by injection molding or casting.-   45. A method of manufacturing a detection disc as clauseed in    clauses 42, 43 or 44 comprising providing the disc with features as    described in any of clauses 15 to 30.-   46. A method of finding a focal plane in an apparatus for detection    of microscopic objects in a fluid, wherein the method comprises:    -   providing an optically transparent substrate having a focus        structure, the focus structure comprising a groove with sloping        sidewalls provided in a first surface of the substrate;    -   illuminating the substrate from a second surface, opposed to the        first surface;    -   imaging the focus structure on the first surface using an        imaging apparatus, and    -   focussing the imaging apparatus on the focus structure,    -   wherein the angle between each sidewall of the focus structure        and the first surface is greater than the critical angle of the        substrate.-   47. A method according to clause 46 wherein the focus structure has    a V-shaped cross-section.-   48. A method according to clause 46 or 47, further comprising using    information about the focal plane found by the imaging apparatus to    focus a further, separate, imaging apparatus to the same focal    plane.-   49. A method according to clause 48, wherein the focussing imaging    apparatus is an area detector and the further imaging apparatus is a    line detector.-   50. A method according to any of clauses 46 to 49, comprising    illuminating the detection channel with a line beam, the projection    of the line beam being co-linear to the line of the line detector at    the imaging plane.-   51. A method according to any of clauses 46 to 50 wherein the    substrate is a detection disc as described in any of clauses 15 to    30 and the focus structure is a focus structure of the disc.-   52. A method of focussing a first imaging apparatus to a focal plane    in an apparatus for detection of microscopic objects in a fluid,    wherein the method comprises:    -   imaging a focus structure with a second imaging apparatus,    -   focussing the second imaging apparatus on the focus structure to        find the focal plane; and    -   focussing the first imaging apparatus to the focal plane to        which the second imaging apparatus is focussed.-   53. A method according to clause 52, wherein the first imaging    apparatus is a line detector, and the second imaging apparatus is an    area detector.-   54. A method according to clause 52, wherein the first imaging    apparatus is an area detector in which a selected line or a selected    subset of lines can be read out.-   55. A method according to clause 52, wherein a single line detector    serves as both the first and second imaging apparatus.-   56. A method according to clause 52, wherein a single imaging    apparatus where a selected line or a selected subset of lines can be    read out serves the purpose of both the first and second imaging    apparatus.-   57. A method according to any of clauses 52 to 56, wherein the focus    structure comprises a groove with sloping sidewalls provided in a    first surface of the substrate, and wherein the angle between each    sidewall of the focus structure and the first surface is greater    than the critical angle of the substrate.-   58. A method according to any of clauses 52 to 57, comprising    illuminating the detection channel with a line beam, the projection    of the line beam being co-linear to the line of the line detector at    the imaging plane-   59. A method according to any of clauses 52 to 58, wherein the    method comprises using a detection disc as described in any of    clauses 15 to 30 and the focus structure is a focus structure of the    disc.-   60. An apparatus for detecting and/or counting microscopic objects    comprising:    -   a detection disc comprising a detection channel for carrying the        microscopic objects in a sample fluid and a separate focus        structure associated with the detection channel,    -   a first imaging apparatus for finding a focal plane by focussing        on the focus structure; and    -   a second imaging apparatus for detecting and/or counting the        objects in the detection channel, the second imaging apparatus        being focussed to the focal plane using information about the        focal plane from the first imaging apparatus.-   61. An apparatus according to clause 60, wherein the first imaging    apparatus is a line detector, and the second imaging apparatus is an    area detector.-   62. An apparatus according to clause 60, wherein the first imaging    apparatus is an area detector in which a selected line or a selected    subset of lines can be read out.-   63. An apparatus according to clause 62 wherein the area detector is    a CMOS area detector-   64. An apparatus according to clause 60, wherein a single line    detector serves as both the first and second imaging apparatus.-   65. An apparatus according to clause 60, wherein a single imaging    apparatus where a selected line or a selected subset of lines can be    read out serves the purpose of both the first and second imaging    apparatus.-   66. An apparatus according to any of clauses 60 to 65, wherein the    focus structure comprises a groove with sloping sidewalls provided    in a first surface of the substrate, and wherein the angle between    each sidewall of the focus structure and the first surface is    greater than the critical angle of the substrate.-   67. An apparatus according to clause any of clauses 60 to 66,    comprising a light source for illuminating the detection channel    with a line beam, the projection of the line beam being co-linear to    the line of the line detector at the imaging plane-   68. An apparatus according to clause any of clauses 60 to 67,    wherein the detection disc is as described in any of clauses 15 to    30.-   69. An apparatus for detecting and/or counting microscopic objects    comprising:    -   a detection disc comprising a detection channel for carrying the        microscopic objects in a sample fluid and a separate focus        structure associated with the detection channel,    -   a first imaging apparatus for finding a focal plane by focussing        on the focus structure;    -   a second imaging apparatus for counting the objects in a        detection channel, the second imaging apparatus being focussed        to the focal plane using information about the focal plane from        the first imaging apparatus,    -   wherein the focus structure comprises a groove with sloping        sidewalls provided in a first surface of the detection disc, and        wherein the angle between each sidewall of the focus structure        and the first surface is greater than the critical angle of the        substrate.-   70. An apparatus according to clause 69 wherein the focus structure    has a V-shaped cross-section.-   71. An apparatus according to clause 69 or 70, comprising a further,    separate, imaging apparatus that receives information about the    focal plane found by the focussing imaging apparatus and uses this    information to focus on the focal plane.-   72. An apparatus according to clause 71, wherein the focussing    imaging apparatus is an area detector and the further imaging    apparatus is a line detector.-   73. An apparatus according to clause 72, comprising a light source    for illuminating the detection channel with a line beam, the    projection of the line beam being co-linear to the line of the line    detector at the imaging plane.-   74. An apparatus according to any of clauses 69 to 73 wherein the    detection disc is as described in any of clauses 15 to 30.-   75. A sample processing module for an apparatus for detecting and/or    counting microscopic objects, the sample processing module    comprising: a carousel for holding a plurality of sample containers;    a number of stations for sample processing steps including a filling    station for filling the sample containers, a heating station for    heating a sample in the sample container; a cooling station for    cooling the sample in the sample container; an agitation station for    agitating the sample in the sample container; and a purging station    for purging the sample from the sample container and passing it to a    detection module for analysis of the sample; wherein the heating and    cooling stations act on the sample without contact with the sample.-   76. A sample processing module according to clause 75, wherein the    heating station uses infra-red or heated air to heat the sample.-   77. A sample processing module according to clause 75 or 76, wherein    each of the filling, heating, cooling and purging stations act    without contact between the stations and the sample container held    in the carousel.-   78. A sample processing module according to clause 75, 76 or 77,    wherein the filling station comprises a dispensing nozzle arranged    to fill the sample container without contact with the container or    carousel.-   79. A sample processing module according to any of clauses 75 to 79,    wherein the purging station comprises a compressed air source    arranged to expel the sample through a hole in the container without    contact between the compressed air source and the container or    carousel.

What is claimed is:
 1. A method of manufacturing a detection disc forcarrying samples in an apparatus for detection of microscopic objects ina fluid, the method comprising: providing a crystallographic siliconsubstrate; etching a first channel into a (100) plane of thecrystallographic silicon substrate; etching a separate first focusstructure into the (100) plane of the crystallographic silicon substrateby wet etching, the first focus structure comprising a groove havingsloping sidewalls which are aligned with {111} planes of thecrystallographic silicon substrate; producing a production master plateusing the crystallographic silicon substrate; and injection molding thedetection disc using the production master plate, the detection discincluding a detection channel and a separate second focus structure, thedetection channel having a same geometry as the first channel and thesecond focus structure having a same geometry as the first focusstructure in the crystallographic silicon substrate, and wherein thedetection disc comprises a material having a refractive index of greaterthan 1.22.
 2. A method according to claim 1 wherein the first focusstructure has a V-shaped cross-section.
 3. A method according to claim1, wherein a depth of the first focus structure is less than a depth ofthe first channel.
 4. A method according to claim 1, wherein an anglebetween each sidewall of the second focus structure and the firstsurface of the detection disc is greater than a critical angle of thematerial of the detection disc.
 5. A method according to claim 4,wherein the angle between each sidewall of the second focus structureand the first surface of the detection disc is 54.7°.
 6. A methodaccording to claim 1, wherein an angle between the (100) plane of thecrystallographic silicon substrate and the {111} planes of thecrystallographic silicon substrate is 54.7°.
 7. A method according toclaim 1, wherein the production master plate has an opposite geometricpolarity from the crystallographic silicon substrate.
 8. A method ofmanufacturing a substrate for use in manufacturing a production masterplate for use in manufacturing a detection disc comprising a materialhaving a refractive index of greater than 1.22 and being for carryingsamples in an apparatus for detection of microscopic objects in a fluid,the substrate including a first channel and a separate first focusstructure, for forming a corresponding detection channel and acorresponding separate second focus structure in the detection disc, themethod comprising: providing the substrate having a crystallographicsilicon structure including crystalline planes; etching the firstchannel into a (100) plane of the crystallographic silicon substratewith an orientation independent of an orientation of the crystallineplanes; and wet etching the first focus structure into the (100) planeof the crystallographic silicon substrate with an orientation alignedwith one of the crystalline planes, wherein the first focus structure isa groove having sloping sidewalls, which are aligned with {111} planesof the crystallographic silicon substrate.
 9. A method according toclaim 8 wherein a depth of the first focus structure is less than adepth of the first channel.
 10. A method according to claim 8, whereinan angle between the (100) plane of the crystallographic siliconsubstrate and the {111} planes of the crystallographic silicon substrateis 54.7°.
 11. A method of manufacturing a detection disc for carryingsamples in an apparatus for detection of microscopic objects in a fluid,the method comprising: providing a crystallographic silicon substrate;etching a first channel into a (100) plane of the crystallographicsilicon substrate; etching a separate first focus structure into the(100) plane of the crystallographic silicon substrate by wet etching,the first focus structure comprising a groove having sloping{111}-oriented sidewalls, wherein an angle between the (100) plane ofthe crystallographic silicon substrate and a {111} plane is 54.7°;producing a production master plate using the crystallographic siliconsubstrate; and injection molding the detection disc using the productionmaster plate, the detection disc including a detection channel and aseparate second focus structure, the detection channel having a samegeometry as the first channel and the second focus structure having asame geometry as the first focus structure in the crystallographicsilicon substrate, and wherein the detection disc comprises a materialhaving a refractive index of greater than 1.22 to give rise to totalinternal reflection from the sidewalls when a collimated light beamilluminates the sidewalls of the second focus structure from below.